1. Field of the Invention
This invention relates generally to analytical determinations of peroxidatively active substances in test samples, and particularly to a composition, test means, device and method useful in such determinations and resistant to possible adverse effects from ascorbic acid which may also be present in the sample.
2. Background Art
Many analytical methods are presently available for detecting the presence of peroxidatively active substances in biological samples such as urine, fecal suspensions, and gastrointestinal contents. For example, hemoglobin and its derivatives, the analytes determined by conventional occult blood tests, are typical of peroxidatively active substances because they behave in a manner similar to the enzyme peroxidase; as such, they are also referred to as pseudoperoxidases. Peroxidatively active substances are enzyme-like by virtue of their catalysis of the redox reaction between peroxides or hydroperoxides and such indicator compounds as benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene and the like, producing a detectable response such as a color change. Hence, most methods for determining the presence of occult blood in test samples rely on this pseudoperoxidase activity.
A number of analytical methods for determining peroxidatively active substances have evolved which rely on the enzyme-like catalysis of the peroxidative oxidation of colorforming indicators. Primarily, these include wet chemistry or solution procedures and the so-called "dip-and-read" type, reagent-bearing strip devices. Of the former, a typical example is set forth in R. M. Henry, et al., Clinical Chemistry Principles and Techniques, 2nd ed., 1124-1125 (Hagerstown, Md.: Harper and Row, 1974). This exemplary procedure involves the use of glacial acetic acid (buffer), diphenylamine (indicator) and hydrogen peroxide. While such wet chemistry methods have proven analytical utility, they possess many disadvantages, two examples of which are poor reagent stability and inadequate sensitivity.
Another method for the determination of peroxidatively active substances, and one presently preferred by most clinical analysts, utilizes the so-called "dip-and-read" reagent strip device. Typical of such "dip-and-read" devices is one commercially available from the Ames Division of Miles Laboratories, Inc. under the trademark HEMASTIX.RTM.. This device comprises a porous paper matrix impregnated with a buffered mixture of an organic hydroperoxide and an indicator, affixed to a plastic strip or handle. Upon immersion of the matrix in a liquid containing hemoglobin, myoglobin, erythrocytes or other peroxidatively active substances, i.e., pseudoperoxidases, a blue color develops in the matrix, the intensity of which is proportional to the concentration of the substance in the sample. By comparing the color developed in the matrix to a standard color chart, the analyst can determine, on a semiquantitative basis, the amount of analyte present in the sample.
Primarily, the advantages of such reagent strips over wet chemistry methods are: (1) the strip format is easier to use, requiring neither the preparation of reagents nor attendant apparatus; and (2) greater stability of reagents is afforded in the strip, resulting in improved accuracy, sensitivity and economy.
Whether a particular analysis for a peroxidatively active species is undertaken by either of the aforedescribed methods, a problem inherent to both exists: interference caused by the presence in the sample of reducing agents in general and ascorbic acid or ascorbate ion in particular (hereafter referred to as ascorbate interference). In the case of urinalysis for example, the recent popularity of diets which include high dosages of vitamin C (ascorbic acid) has resulted in serious ascorbate interference problems in analyzing for certain urine constituents, such as occult blood. Patients on such diets typically exhibit elevated levels of urinary ascorbate.
As early as 1938, the adverse effects of reducing agents such as ascorbate were recognized. R. Kohn and R. M. Watrous, Journal of Biological Chemistry, 124, 163-168 (1938). The same problem still plagues this area of diagnostic analysis, as evidenced by a proposal of 1979 that when an occult blood (a pseudoperoxidase) analysis is performed a simultaneous ascorbate analysis should also be performed in order to gauge the accuracy of the occult blood determination. L. Nielsen, P. J. Jorgensen and A. C. Hansen, Ugeskrift for Laeger, 141, 791-793 (1979).
Many attempts at removing ascorbate interference with test systems, such as systems containing glucose-sensitive reagents, are reported in the literature. With regard to glucose-sensitive assays, approaches have ranged from filtering out ascorbate before it reaches the reagents, to the utilization of an enzyme to decompose it, in situ.
Accordingly, Canadian Pat. No. 844,564 to Dahlqvist discloses a device for glucose determination in urine or other media which includes, in addition to a porous portion impregnated with normal glucose-responsive reagents, an additional portion to receive the urine test sample. The sample-receiving portion comprises an ion exchange material, whose singular function in the device is to adsorb any ascorbate present in the urine sample.
U.S. Pat. No. 4,168,205 to Danninger et al., suggests incorporating the enzyme ascorbate oxidase into the test reagent formulation; any ascorbate present in the sample will be enzymatically oxidized by the ascorbate oxidase to dehydroascorbate, a compound which does not adversely affect the desired analysis.
Another approach to alleviating ascorbate interference is reflected in Japanese Provisional Patent Publication No. 55757/1983 to Fuji Zoki Seiyaku K.K. The publication discloses the use of metal chelates of various ligands such as ethylenediaminetetracetic acid and diethylenetriaminepentacetic acid to pretreat a sample which will then be assayed for cholesterol, glucose or other components such as uric acid.
U.S. Pat. No. 3,411,887 to Ku describes the elimination of ascorbate interference with reagent systems which rely on enzymatic oxidizing substances such as glucose oxidase, by using an ascorbate "trapping system". The "trapping system" involves an ionizable heavy metal compound which when ionized has an oxidation-reduction potential falling between a redox indicator dye and ascorbate. Some suitable metals which are cited as examples include cobalt, iron, mercury and nickel.
U.S. Pat. No. 4,288,541 to Magers et al., commonly assigned herewith, describes the use of mercuric ion complexes, such as mercuric sarcosinate, to impart ascorbate resistance to a glucose/glucose oxidase assay system.
In addition to the foregoing, attention to the ascorbate problem with glucose tests is manifested by:
1. H. Gifford, et al., J. Amer. Med. Assoc., 178, 149-150 (1961). PA0 2. P. O'Gorman, et al., Brit. Med. J., 603-606 (1960). PA0 3. R. Brandt, et al., Clin. Chem. Acta, 51, 103-104 (1974). PA0 4. R. Brandt, et al., Am. J. Clin. Pathol., 68, 592-594 (1977).
Similar to the approach of the above-cited Ku patent, other literature deals with the complexing and oxidation of ascorbate using cobalt. For example, G. Bragagnolo, Ann. Chim. Applicata, 31, 350-368, 1941, reported that solutions of ascorbic acid were oxidized by air in the presence of cobalt metal. Also, similar activity has been reported for Co(NH.sub.3).sub.6 Cl.sub.3 by Tomokichi Iwasaki in Journal of the Chemical Society of Japan, 63, 820-826 (1942).
Although the foregoing art deals extensively with analytical systems for glucose determinations, no suggestions are set forth as to resolution of the ascorbate interference problem in connection with the determination of such peroxidatively active substances as peroxidase and pseudoperoxidases such as occult blood (hemoglobin). Notwithstanding the disclosure of the Ku patent, supra, the aforementioned art indicates that metal ions, such as Co(III), are, in fact, also pseudoperoxidases. For example, Co(III) acetate is used commercially to catalytically decompose cumene hydroperoxide. [The Merck Index, 9th ed., 311 (1976).] A series of Co(III) complexes are reported to catalytically decompose peroxides by K. Lohs., Monatsber. Deut. Akad. Wiss. Berlin, 8, 657-659 (1966) (See Chem. Abstracts, 67, 120383z. 1967). One skilled in the art would clearly, therefore, be led to believe that the use of any such metal complexes in a typical analytical formulation for the determination of peroxidatively active substances, i.e., one containing an organic hydroperoxide and an indicator, would cause deleterious interaction with the hydroperoxide, either producing "false positive" results, or otherwise rendering it unreactive to the peroxidatively active substance of interest, such as occult blood, and thus useless for such determinations. In fact, efforts to use mercuric complexes, such as mercuric sarcosinate, in occult blood tests failed.
U.S. Pat. No. 4,310,626 to Burkhardt et al., commonly assigned herewith, addresses the foregoing problem in describing the use of ammonium Co(III) complexes for abating ascorbate interference with compositions for determining peroxidatively active substances. This patent discloses such compositions which comprise an organic hydroperoxide and a suitable indicator, such as 3,3'5,5'-tetramethylbenzidine, together with ammonium Co(III) complexes such as Co(NH.sub.3).sub.6 Cl.sub.3, among others. These complexes, however did not impart sufficient ascorbate-resistance to an occult blood test to be commercially advantageous.
Other approaches to dealing with ascorbate interference in analytical determinations of peroxidatively active substances include, for example, West German Pat. No. 29 07 628. This German patent involves urinalysis in solution, whereby a urine sample is pretreated with one or more oxidants to remove ascorbate, and then contacted by the appropriate analytical reagents. The oxidants disclosed are sodium iodate, sodium periodate, calcium hydrochlorite, potassium triiodide, sodium hydrochlorite, chloroamine and bromosuccinimide.
In summary, various approaches to alleviating the interference problem presented by ascorbic acid in determinaton of peroxidatively active substances have included such techniques as the use of various Co(III) ammonium complexes, pretreatment of the sample with oxidizing agents and direct addition to the reagent composition of alkali metal iodates.
Pseudoperoxidases such as hemoglobin are often studied as alternate peroxidase systems in order to learn more about the mechanism of action of natural peroxidases such as those obtained from horseradish or potatoe sources. Ascorbic acid has long been known as a classical substrate for peroxidase, and ascorbic acid oxidation in the presence of metal chelates which act as pseudoperoxidases is a known phenomenon. In 1967 and 1968, M. Khan and A. Martell reported on kinetic studies of ascorbic acid oxidation in the presence of several ferric and cupric chelates over a pH range of 1.8 to 3.45. [Khan, M. and Martell, A., J. Am. Chem. Soc., 89, 4176 (1967); J. Am. Chem. Soc., 89, 7104 (1967); J. Am. Chem. Soc., 90, 3386 (1968).] A variety of kinetic and thermodynamic parameters were investigated in these studies. The result was a rank order of effectiveness of different chelates according to their abilities to oxidize ascorbic acid. Of four aminopolycarboxylic acids studied by these writers, the N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) chelate of Fe.sup.++ was found to be the fastest oxidant. Both the work of Martell, and an earlier study by Grinstead, regard this ascorbic acid oxidation activity to constitute a "model" peroxidase system. [Grinstead, R. R., J. Am. Chem. Soc., 82, 3464 (1960).] Such studies as the foregoing by Grinstead constituted an attempt to study the peroxidase mechanism by means of a certain ferric chelate whose structure could mimic that of the iron-containing heme found at the active site of the enzyme peroxidase. Indeed, the writers use the phrase "model peroxidase system" repeatedly in their papers. However, because of the "model" peroxidase activity shown by this chelate and others which are similar in reactivity, one would certainly not expect that such substances could be incorporated into organic hydroperoxide/indicator systems such as those now typically used in analytical reagent compositions and devices for the determination of peroxidase or other peroxidatively active substances. Moreover, research studies undertaken by the assignee of the present invention in connection with peroxidase activity revealed that, with indicators such as 3,3',5,5'-tetramethylbenzidine (TMB) or o-tolidine (indicators which are typically used in analytical systems to determine the presence of peroxidatively active substances), such activity could be expected to occur some 200 times faster than with ascorbic acid. White-Stevens, R. H., Clin. Chem., 28, 578 (1982). Accordingly, it can be assumed that if such peroxidatively active metal chelates, "model" peroxidases, act to so readily oxidize ascorbic acid--an assumption made by Khan, Martell and Grinstead--then the peroxidase reaction with such indicators as TMB would proceed at least at the same rate as with ascorbic acid, if not some 200 times faster (as suggested in these latter studies, which were undertaken on horseradish peroxidase). Clearly, if an extremely reactive analyte is incorporated into the very reagent formulation designed to change color in the presence of that analyte, it is to be expected that "false positive" results would be obtained.